PDGF function in diverse neural crest cell populations

Activation of platelet derived growth factor (PDGF) receptors causes context-dependent cellular responses, including proliferation and migration, and studies in model organisms have demonstrated that this receptor family (PDGFRα and PDGFRβ) is required in many mesenchymal and migratory cell populations during embryonic development. One of these migratory cell populations is the neural crest, which forms cranial bone and mesenchyme, sympathetic neurons and ganglia, melanocytes, and smooth muscle. Mice with disruption of PDGF signaling exhibit defects in some of these neural crest derivatives including the palate, aortic arch, salivary gland, and thymus. Although many of these neural crest defects were identified many years ago, the mechanism of action of PDGF in neural crest remains controversial. In this review, we examine the current knowledge of PDGF function during neural crest cell (NCC) development, focusing on its role in the formation of different neural crest-derived tissues and the implications for PDGF receptors in NCC-related human birth defects.     

Platelet-derived growth factor receptors direct vascular development independent of vascular smooth muscle cell function

Complete loss of platelet-derived growth factor (PDGF) receptor signaling results in embryonic lethality around embryonic day 9.5, but the cause of this lethality has not been identified. Because cardiovascular failure often results in embryonic lethality at this time point, we hypothesized that a failure in cardiovascular development could be the cause. To assess the combined role of PDGF receptor α (PDGFRα) and PDGFRβ, we generated embryos that lacked these receptors in cardiomyocytes and vascular smooth muscle cells (VSMC) using conditional gene ablation. Deletion of either PDGFRα or PDGFRβ caused no overt vascular defects, but loss of both receptors using an SM22α-Cre transgenic mouse line led to a disruption in yolk sac blood vessel development. The cell population responsible for this vascular defect was the yolk sac mesothelial cells, not the cardiomyocytes or the VSMC. Coincident with loss of PDGF receptor signaling, we found a reduction in collagen deposition and an increase in MMP-2 activity. Finally, in vitro allantois cultures demonstrated a requirement for PDGF signaling in vessel growth. Together, these data demonstrate that PDGF receptors cooperate in the yolk sac mesothelium to direct blood vessel maturation and suggest that these effects are independent of their role in VSMC development.     

A Critical Role for PDGFRα Signaling in Medial Nasal Process Development

The primitive face is composed of neural crest cell (NCC) derived prominences. The medial nasal processes (MNP) give rise to the upper lip and vomeronasal organ, and are essential for normal craniofacial development, but the mechanism of MNP development remains largely unknown. PDGFRα signaling is known to be critical for NCC development and craniofacial morphogenesis. In this study, we show that PDGFRα is required for MNP development by maintaining the migration of progenitor neural crest cells (NCCs) and the proliferation of MNP cells. Further investigations reveal that PI3K/Akt and Rac1 signaling mediate PDGFRα function during MNP development. We thus establish PDGFRα as a novel regulator of MNP development and elucidate the roles of its downstream signaling pathways at cellular and molecular levels.     

Comprehensive Dissection of PDGF-PDGFR Signaling Pathways in PDGFR Genetically Defined Cells

Despite the growing understanding of PDGF signaling, studies of PDGF function have encountered two major obstacles: the functional redundancy of PDGFRα and PDGFRβ in vitro and their distinct roles in vivo. Here we used wild-type mouse embryonic fibroblasts (MEF), MEF null for either PDGFRα, β, or both to dissect PDGF-PDGFR signaling pathways. These four PDGFR genetically defined cells provided us a platform to study the relative contributions of the pathways triggered by the two PDGF receptors. They were treated with PDGF-BB and analyzed for differential gene expression, in vitro proliferation and differential response to pharmacological effects. No genes were differentially expressed in the double null cells, suggesting minimal receptor-independent signaling. Protean differentiation and proliferation pathways are commonly regulated by PDGFRα, PDGFRβ and PDGFRα/β while each receptor is also responsible for regulating unique signaling pathways. Furthermore, some signaling is solely modulated through heterodimeric PDGFRα/β.     

G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis

The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development.     

A Reactive Oxygen Species-Mediated,Self-Perpetuating Loop Persistently Activates Platelet-Derived Growth Factor Receptor α

The platelet-derived growth factor (PDGF) receptors (PDGFRs) are central to a spectrum of human diseases. When PDGFRs are activated by PDGF, reactive oxygen species (ROS) and Src family kinases (SFKs) act downstream of PDGFRs to enhance PDGF-mediated tyrosine phosphorylation of various signaling intermediates. In contrast to these firmly established principles of signal transduction, much less is known regarding the recently appreciated ability of ROS and SFKs to indirectly and chronically activate monomeric PDGF receptor α (PDGFRα) in the setting of a blinding condition called proliferative vitreoretinopathy (PVR). In this context, we made a series of discoveries that substantially expands our appreciation of epigenetic-based mechanisms to chronically activate PDGFRα. Vitreous, which contains growth factors outside the PDGF family but little or no PDGFs, promoted formation of a unique SFK-PDGFRα complex that was dependent on SFK-mediated phosphorylation of PDGFRα and activated the receptor''s kinase activity. While vitreous engaged a total of five receptor tyrosine kinases, PDGFRα was the only one that was activated persistently (at least 16 h). Prolonged activation of PDGFRα involved mTOR-mediated inhibition of autophagy and accumulation of mitochondrial ROS. These findings reveal that growth factor-containing biological fluids, such as vitreous, are able to tirelessly activate PDGFRα by engaging a ROS-mediated, self-perpetuating loop.     

Cooperation between the PDGF receptors in cardiac neural crest cell migration

Neural crest cells (NCCs) are essential components of the sympathetic nervous system, skin, craniofacial skeleton, and aortic arch. It has been known for many years that perturbation of migration, proliferation, and/or differentiation of these cells leads to birth defects such as cleft palate and persistent truncus arteriosus (PTA). Previously, we had shown that disruption of the platelet-derived growth factor receptor (PDGFR) alpha in NCCs resulted in defects in craniofacial and aortic arch development, the latter with variable penetrance. Because we observed ventricular septal defects in embryos that are null for the PDGFRbeta, we hypothesized that both PDGF receptors are involved in NCC formation. Here, we show that both receptors are expressed in cardiac NCCs and that the combined loss of the PDGFRalpha and PDGFRbeta in NCCs resulted in NCC-related heart abnormalities, including PTA and a ventricular septal defect (VSD). Using NCC lineage tracing, we observed that loss of PDGF receptor signaling resulted in reduced NCCs in the conotruncus region, leading to defects in aortic arch septation. These results indicate that while PDGFRalpha plays a predominant role in NCC development, the PDGFRbeta is expressed by and functions in cardiac NCCs. Combined PDGF receptor signaling is required for sufficient recruitment of cardiac NCCs into the conotruncal region and for formation of the aortico-pulmonary and ventricular septum.     

Platelet-derived Growth Factor β-Receptor,Transforming Growth Factor β Type I Receptor,and CD44 Protein Modulate Each Other's Signaling and Stability

Growth factors, such as platelet-derived growth factor BB (PDGF-BB) and transforming growth factor β (TGFβ), are key regulators of cellular functions, including proliferation, migration, and differentiation. Growth factor signaling is modulated by context-dependent cross-talk between different signaling pathways. We demonstrate in this study that PDGF-BB induces phosphorylation of Smad2, a downstream mediator of the canonical TGFβ pathway, in primary dermal fibroblasts. The PDGF-BB-mediated Smad2 phosphorylation was dependent on the kinase activities of both TGFβ type I receptor (TβRI) and PDGF β-receptor (PDGFRβ), and it was prevented by inhibitory antibodies against TGFβ. Inhibition of the activity of the TβRI kinase greatly reduced the PDGF-BB-dependent migration in dermal fibroblasts. Moreover, we demonstrate that the receptors for PDGF-BB and TGFβ interact physically in primary dermal fibroblasts and that stimulation with PDGF-BB induces internalization not only of PDGFRβ but also of TβRI. In addition, silencing of PDGFRβ by siRNA decreased the stability of TβRI and delayed TGFβ-induced signaling. We further show that the hyaluronan receptor CD44 interacts with both PDGFRβ and TβRI. Depletion of CD44 by siRNA increased signaling via PDGFRβ and TβRI by stabilizing the receptor proteins. Our data suggest that cross-talk between PDGFRβ and TβRI occurs in dermal fibroblasts and that CD44 negatively modulates signaling via these receptors.     

A PDGF receptor mutation in the mouse (Patch) perturbs the development of a non-neuronal subset of neural crest-derived cells.

The Patch (Ph) mutation in mice is a deletion of the gene encoding the platelet-derived growth factor receptor alpha subunit (PDGFR alpha). Patch is a recessive lethal recognized in heterozygotes by its effect on the pattern of neural crest-derived pigment cells, and in homozygous mutant embryos by visible defects in craniofacial structures. Since both pigment cells and craniofacial structures are derived from the neural crest, we have examined the differentiation of other crest cell-derived structures in Ph/Ph mutants to assess which crest cell populations are adversely affected by this mutation. Defects were found in many structures populated by non-neuronal derivatives of cranial crest cells including the thymus, the outflow tract of the heart, cornea, and teeth. In contrast, crest-derived neurons in both the head and trunk appeared normal. The expression pattern of PDGFR alpha mRNA was determined in normal embryos and was compared with the defects present in Ph/Ph embryos. PDGFR alpha mRNA was expressed at high levels in the non-neuronal derivatives of the cranial neural crest but was not detected in the crest cell neuronal derivatives. These results suggest that functional PDGF alpha is required for the normal development of many non-neuronal crest-derived structures but not for the development of crest-derived neuronal structures. Abnormal development of the non-neuronal crest cells in Ph/Ph embryos was also correlated with an increase in the diameter of the proteoglycan-containing granules within the crest cell migratory spaces. This change in matrix structure was observed both before and after crest cells had entered these spaces. Taken together, these observations suggest that functional PDGFR alpha can affect crest development both directly, by acting as a cell growth and/or survival stimulus for populations of non-neurogenic crest cells, and indirectly, by affecting the structure of the matrix environment through which such cells move.     

Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor α and Yes Kinase

There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.     

Stimulation of Platelet-derived Growth Factor Receptor β (PDGFRβ) Activates ADAM17 and Promotes Metalloproteinase-dependent Cross-talk between the PDGFRβ and Epidermal Growth Factor Receptor (EGFR) Signaling Pathways

Binding of the platelet-derived growth factor (PDGF)-B to its receptor PDGFRβ promotes proliferation, migration, and recruitment of pericytes and smooth muscle cells to endothelial cells, serving to stabilize developing blood vessels. The main goals of this study were to determine whether the extracellular domain of the PDGFRβ can be proteolytically released from cell membranes and, if so, to identify the responsible sheddase and determine whether activation of the PDGFRβ stimulates its shedding and potentially that of other membrane proteins. We found that the PDGFRβ is shed from cells by a metalloproteinase and used loss-of-function experiments to identify ADAM10 as the sheddase responsible for constitutive and ionomycin-stimulated processing of the PDGFRβ. Moreover, we showed that ligand-dependent activation of the PDGFRβ does not trigger its own shedding by ADAM10, but instead it stimulates ADAM17 and shedding of substrates of ADAM17, including tumor necrosis factor α and transforming growth factor α. Finally, we demonstrated that treatment of mouse embryonic fibroblasts with PDGF-B triggers a metalloproteinase-dependent cross-talk between the PDGFRβ and the epidermal growth factor receptor (EGFR)/ERK1/2 signaling axis that is also critical for PDGF-B-stimulated cell migration, most likely via ADAM17-dependent release and activation of ligands of the EGFR. This study identifies the principal sheddase for the PDGFRβ and provides new insights into the mechanism of PDGFRβ-dependent signal transduction and cross-talk with the EGFR.     

Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

Background

Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization.

Methodology/Principal Findings

In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels.

Significance/Conclusion

Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.     

Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.     

The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

Background

The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule.

Methodology/Principal Findings

To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis.

Conclusions/Significance

Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.     

Targeted mutagenesis on PDGFRα-Fc identifies amino acid modifications that allow efficient inhibition of HCMV infection while abolishing PDGF sequestration

Platelet-derived growth factor receptor alpha (PDGFRα) serves as an entry receptor for the human cytomegalovirus (HCMV), and soluble PDGFRα-Fc can neutralize HCMV at a half-maximal effective concentration (EC50) of about 10 ng/ml. While this indicates a potential for usage as an HCMV entry inhibitor PDGFRα-Fc can also bind the physiological ligands of PDGFRα (PDGFs), which likely interferes with the respective signaling pathways and represents a potential source of side effects. Therefore, we tested the hypothesis that interference with PDGF signaling can be prevented by mutations in PDGFRα-Fc or combinations thereof, without losing the inhibitory potential for HCMV. To this aim, a targeted mutagenesis approach was chosen. The mutations were quantitatively tested in biological assays for interference with PDGF-dependent signaling as well as inhibition of HCMV infection and biochemically for reduced affinity to PDGF-BB, facilitating quantification of PDGFRα-Fc selectivity for HCMV inhibition. Mutation of Ile 139 to Glu and Tyr 206 to Ser strongly reduced the affinity for PDGF-BB and hence interference with PDGF-dependent signaling. Inhibition of HCMV infection was less affected, thus increasing the selectivity by factor 4 and 8, respectively. Surprisingly, the combination of these mutations had an additive effect on binding of PDGF-BB but not on inhibition of HCMV, resulting in a synergistic 260fold increase of selectivity. In addition, a recently reported mutation, Val 242 to Lys, was included in the analysis. PDGFRα-Fc with this mutation was fully effective at blocking HCMV entry and had a drastically reduced affinity for PDGF-BB. Combining Val 242 to Lys with Ile 139 to Glu and/or Tyr 206 to Ser further reduced PDGF ligand binding beyond detection. In conclusion, this targeted mutagenesis approach identified combinations of mutations in PDGFRα-Fc that prevent interference with PDGF-BB but maintain inhibition of HCMV, which qualifies such mutants as candidates for the development of HCMV entry inhibitors.     

Cytoplasmic protein methylation is essential for neural crest migration

As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration.     

Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1^Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures.     

PDGF Receptor-α Does Not Promote HCMV Entry into Epithelial and Endothelial Cells but Increased Quantities Stimulate Entry by an Abnormal Pathway

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128–131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα–transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128–131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128–131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types.     

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.